Doubled haploid (DH) populations, particularly those from subspecies crosses possessing the wide compatible gene S5ⁿ, are important germplasm resources for rice genetic studies and breeding, but their feature and potential have not been fully assessed and explored. In the present study, we produced a DH population from the hybrid of japonica 668B and wide compatible indica T23. Genotyping of the S5 locus with allele-specific markers for ORF3, ORF4 and ORF5 revealed a potential recombination hot spot in the ORF3-ORF4 region. Haplotyping analysis revealed that 21/34 subspecies specific Indel markers segregated in distortion in the DH population, with a few lines having indica alleles either extremely low (1.7%) or high (98.3%), with little effect of the S5 allele. While DH lines with the S5ⁿ allele had higher frequency of indica alleles, no effect of the S5ⁿ allele was observed on all agronomic traits but flowering time. Taken together, the present study advanced understanding of the genetics of wide crosses in general, and DH production in particular between the two rice subspecies, and the new DH population generated will become a useful resource for rice genetic study and breeding in the future.